The present invention relates to a gene involved in actin dynamics and phospholipid hydrolysis in the yeast Aspergillus fumigatus and more particularly to the identification, isolation and cloning of this gene. This invention also relates to a method of using this gene to screen for compounds with antifungal activity.
Profilin is a ubiquitous 12-15 kDa eukaryotic actin and phosphotidylinositol-4,-5-bisphosphate-(PIP2) binding protein. Machesky and Pollard (1993) Trends Cell Biol. 3:381-5. In mammalian cells, the protein functions by binding PIP2 which inhibits the phospholipid hydrolysis by phospholipase C (PLC) in resting cells. Goldschmidt-Clermont et al. (1990) Science 247:1575-8. When cells divide following activation of tyrosine kinase receptors, PLC is phosphorylated and thereby activated to hydrolyze PIP2 even in the presence of profilin. Goldschmidt-Clermont et al. (1991) Science 251:1231-3. This hydrolysis releases the known messengers inositol triphosphate (IP3) and diacylglycerol (DAG) and profilin. Profilin moves from the plasma membrane to the cytosol, where it binds to actin to catalyze the rearrangment of the cytoskeleton. Goldschmidt-Clermont and Jamey (1991) Cell 66:419-21.
Profilin catalyzes release of adenosine diphosphate (ADP) from monomeric or globular (G-) actin. Mockrin et al. (1980) Biochem. 19:5359-62; Goldschmidt-Clermont et al. (1991) J. Cell Biol. 113:1081-9. This activity dramatically increases G-actin""s exchange of ADP for ATP, which deactivates actin for polymerization into microfilaments and faciliatates reorganization of the cytoskeleton. Carlier (1989) Int. Rev. Cytol. 115:139-70. Profilin therefore represents an important component of the eukaryotic cell division cycle as well as, by virtue of its interaction with the actin cytoskeleton, overall cellular integrity, vesicular transport, and cell polarity.
Evidence suggests that profilin is important in the growth cycle of yeasts. Null mutation in the Saccharomyces cerevisiae profilin gene is lethal in many S. cerevisiae strain backgrounds. Magdolen et al. (1988) Mol. Cell Biol. 8:5108-15. In other S. cerevisiae strains, null mutations produce a strain with highly abnormal morphology and severely retarded growth rates. Haarer et al. (1990) J. Cell Biol. 110:105-14. While a role in phospholipid hydrolysis has not been shown, PIP2 dependent translocation of profilin from the plasma membrane into the cytoplasm has been demonstrated. Ostrander et al. (1995) J. Biol. Chem. 46:27045-50. S. cerevisiae profilin has also been shown to interact with the RAS/adenylate cyclase pathway. Vojtek et al. (1991) Cell 66:497-505.
There is a compelling need to prevent and treat systemic fungal infections, many of which are fatal if untreated. Indeed, the 1980s and 1990s witnessed a steep rise in Candida and Aspergillus infections (Musial, CE, Cockerill III, FR, Roberts GD. (1988) Clin Microb Rev 1(4):349-364; Saral R. (1991) Reviews of Infectious Dis 13:487-492). Similar rises in zygomycosis, cryptococcosis, histoplasmosis and fusaria infection have also been noted. The reasons for the rise in fungal infections are several, but a key factor is the growing population of immuno-compromised individuals. This group includes patients with HIV disease (AIDS), older patients, patients who have undergone invasive surgery, transplant patients and burn victims.
As the population of immunosuppressed individuals increases, so do the numbers and types of fungal infections noted in these patients. Although candidiasis remains the most common fungal infection in immunosuppressed patients, aspergillosis, zygomycosis, and other infections by filamentous fungi are a major problem for an increasing number of patients (Georgiev, V. St. (1998) Infectious Diseases in Immunocompromised Hosts, CRC Press, Boca Raton, Fla.; and Fauci, Ark. (1998) Emer Infect Dis. The endemic mycoses, especially histoplasmosis and coccidiodomycosis, also constitute a risk for patients. At particular risk for such infections are those with AIDS, those having undergone bone marrow or organ transplants, those receiving chemotherapy and those who have had debilitating illness, sever injury, prolonged hospitalization, or long-term treatment with antibacterial drugs (NIAID fact sheet, 1996).
According to the CDC""s National Nosocomial Surveillance System, the rate of hospital-related fungal infections nearly doubled between 1980 and 1990. In 1997, an estimated 240,000 individuals showed clinical symptoms of endemic mycoses. With the current approaches to treatment (primarily amphotericin B and the azoles) the mortality rate in patients with systemic fungal infections ranges from 30-100%, depending on the pathogen.
The severity of fungal infections increases as the immune system becomes more dysfunctional. Fungi are among the most ubiquitous pathogens seen in patients with AIDS; virtually all major fungal pathogens cause disease in HIV-positive patients. The majority of untreated HIV-positive patients experience at least one episode of fungal infection and many fungal infections are AIDS-defining illnesses in HIV-infected individuals (Phillips P. (1999).
Therefore, there is a desperate need for new antifungal agents. The recent development of high-throughput screens for the isolation of such agents presents an opportunity for meeting this need. Profilin is amenable to such screening approaches and thus represents an important new target for antifungal drugs.
The present invention concerns an isolated nucleic acid molecule encoding A. fumigatus profilin. Preferably, the A. fumigatus profilin has a sequence that has greater than 70%, 80%, or 90% amino acid sequence identity to SEQ ID NO:2 as measured using a sequence comparison algorithm.
In one aspect, the invention provides an isolated nucleic acid sequence encoding A. fumigatus profilin, wherein the profilin has a sequence that has greater than 70%, 80%, or 90% amino acid sequence identity to SEQ ID NO:2 as measured using a sequence comparison algorithm. In one embodiment, the protein further specifically binds to polyclonal antibodies raised against SEQ ID NO:2.
In one embodiment, the nucleic acid encodes A. fumigatus profilin, or a fragment thereof. In another embodiment, the nucleic acid encodes SEQ ID NO:2. In another embodiment, the nucleic acid has a nucleotide sequence of SEQ ID NO:1.
In one aspect, the nucleic acid comprises a sequence which encodes an amino acid sequence which has greater than 70% sequence identity with SEQ ID NO:2, preferably greater than 80%, more preferably greater than 90%, more preferably greater than 95% or, in another embodiment, has 98 to 100% sequence identity with SEQ ID NO:2.
In one embodiment, the nucleic acid comprises a sequence which has greater than 55 or 60% sequence identity with SEQ ID NO:1, preferably greater than 70%, more preferably greater than 80%, more preferably greater than 90 or 95% or, in another embodiment, has 98 to 100% sequence identity with SEQ ID NO:1. In another embodiment provided herein, the nucleic acid hybridizes under stringent conditions to a nucleic acid having a sequence or complementary sequence of SEQ ID NO:1.
In another aspect, the invention provides an expression vector comprising a nucleic acid encoding A. fumigatus profilin, wherein the protein has a sequence that has greater than 70, 80, or 90% amino acid sequence identity to SEQ ID NO:2 as measured using a sequence comparison algorithm. The invention further provides a host cell transfected with the vector.
In another embodiment, the protein comprises an amino acid sequence of SEQ ID NO:2. In one aspect, the protein provided herein comprises an amino acid sequence which has greater than 70% sequence identity with SEQ ID NO:2, preferably greater than 80%, more preferably greater than 90%, more preferably greater than 95% or, in another embodiment, has 98 to 100% sequence identity with SEQ ID NO:2.
The invention features a substantially purified polypeptide comprising the amino acid sequence of SEQ ID NO:2 or a fragment thereof and more particularly, the ATP hydrolysis pocket or P-loop of the amino acid sequence of SEQ ID NO:2 or a fragment thereof.
Also provided are modulators of the target protein including agents for the treatment of fungal disorders. The agents and compositions provided herein can be used in variety of applications which include the formulation of sprays, powders, and other compositions. Also provided herein are methods of treating fungal disorders.